Date Published: September 8, 2016
Publisher: Public Library of Science
Author(s): Ester Vázquez-Fernández, Matthijn R. Vos, Pavel Afanasyev, Lino Cebey, Alejandro M. Sevillano, Enric Vidal, Isaac Rosa, Ludovic Renault, Adriana Ramos, Peter J. Peters, José Jesús Fernández, Marin van Heel, Howard S. Young, Jesús R. Requena, Holger Wille, Surachai Supattapone.
The structure of the infectious prion protein (PrPSc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc and its N-terminally truncated variant, PrP 27–30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27–30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 Å3, predicted the height of each PrP 27–30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.
Little is known about the structure of the infectious prion protein, the infectious agent causing prion diseases such as sheep and goat scrapie, bovine spongiform encephalopathy or “mad cow disease”, chronic wasting disease in cervids (deer, elk, moose, and reindeer), and Creutzfeldt-Jakob disease in humans. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. The non-infectious, cellular prion protein (PrPC), which has its highest expression levels in neurons, is misfolded through a posttranslational process into an altered, infectious conformer termed PrPSc or prion . The structure of recombinant PrP, which approximates the structure of PrPC, has been solved repeatedly by NMR spectroscopy  and X-ray crystallography . PrPC consists of an unfolded N-terminal domain and a largely α-helical C-terminal domain, which contains three α-helices and a short, two-stranded ß-sheet [2,3]. In contrast, PrPSc has been found by a variety of methods to contain predominantly ß-sheet structure .
Our cryo-EM images of GPI-anchorless PrPSc fibrils, and their subsequent analysis, show that they consist of two intertwined protofilaments, in agreement with a recent study of negatively stained PrPSc fibrils . Each protofilament exhibits an approximately ellipsoidal cross-section with a linear volume of ~9,900 Å3/nm. Given the fact that cryo-EM preserves the native structure of specimens, this information sets a structural restraint for the conformation of GPI-anchorless PrPSc. One important implication is that PrPSc subunits can only fit into protofilaments with the observed dimensions (Table 1), if they are folded up onto themselves. Based on the routine observation of regular 4.8 Å cross-ß signals in individual GPI-anchorless PrP 27–30 fibrils (Fig 3), a ß-solenoid arrangement is the easiest way to accommodate the peptide into the available protofilament volume.